Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis

Authors

  • Hiroya Tomita,

    1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan
    Search for more papers by this author
  • Tadayuki Imanaka,

    1. Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Shiga, Japan
    2. JST, CREST, Tokyo, Japan
    Search for more papers by this author
  • Haruyuki Atomi

    Corresponding author
    1. JST, CREST, Tokyo, Japan
    • Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan
    Search for more papers by this author

For correspondence. E-mail atomi@sbchem.kyoto-u.ac.jp; Tel. (+81) 75 383 2777; Fax (+81) 75 383 2778.

Summary

Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2-oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20–29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk-KPR) displayed reductase activity specific for 2-oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH-dependent enzymes. Tk-KPR activity decreased dramatically in the presence of CoA and KPR activity in cell-free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T. kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea.

Ancillary